“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Somatic embryogenesis inCicer arietinum L: Influence of genotype and auxins

Plant regeneration through somatic embryogenesis was obtained in chickpea (Cicer arietinum L.) using immature cotyledons and immature embryonal axes as explants. 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3.5-6-trichloropicolinic acid (picloram) and 3.6-dichloro-0-anisc, acid (dicamba) in concentrations 1, 2, 5, 10 mg dm^?3 were found better than naphthaleneacetic acid (NAA) (10–20 mg dm^?3) for the induction of globular and heart-shaped somatic embryos. The embryos developed upto the dicotyledonary stage on medium supplemented with saccharose, mannitol and silver nitrate (AgNO₃) and developed further into plantlets on medium containing gibberellic acid (GA₃) and abscisic acid (ABA). The frequency of somatic embryogenesis was dependent on the genotype and auxins used.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Two NAD-dependent alcohol dehydrogenases (E.C. 1.1.1.1) in callus cultures of wheat,rye and triticale

Summary Two NAD-dependent alcohol dehydrogenases ADH-1 and ADH-2, under independent genetic control of genes designated as Adh-1 and Adh-2 located on chromosomes 4A, 4B and 4D, have been reported in aestivum wheat (Hart 1980). Only ADH-1 is expressed in developing seeds, dry seeds, pollen and germinating seedlings. ADH-2 can be induced in seedling roots or shoots under conditions of partial anaerobiosis or by certain chemicals. Expression of ADH-1 and ADH-2 isoenzymes was investigated in undifferentiated calli from aestivum and durum wheats, rye, triticale and also in in vitro regenerated roots and leaves from aestivum cultures. Wheat callus cultures originating from seed, mature and immature embryos, mesocotyl and root, as well as cultures grown on media containing different supplements did not show any variation in the overall expression of ADH-1 or ADH-2, although differences in the band intensities were observed. The callus isoenzyme pattern was similar to that observed in roots under anaerobic conditions. Both ADH-1 and ADH-2 were expressed in in vitro regenerated roots but were absent in regenerated leaves. Expression of ADH-1 and ADH-2 in wheat calli seems to be related to the type of differentiation.     

Callus induction and plant regeneration from immature embryos of rye and triticale

Callus cultures were established from the scutellum, scutellar node and radicle region of immature embryos of rye and octoploid triticale on modified Murashige-Skoog basal medium supplemented with various growth regulators. 2, 4-D, 2, 4, 5-T and 2, 4, 5-Cl, POP were found suitable for initiation and maintenance of callus cultures. Cytokinins had no or inhibitory effect on callus induction and growth. On basal medium containing 5 mg/l of 2,4,5-Cl₃ POP, 16% of triticale and 17% of rye primary cultures exhibited shoot bud regeneration after 3–4 weeks. Transfer of such cultures to basal medium supplemented with zeatin or zeatin in combination with IAA further promoted shoot elongation and plantlet formation. Plantlets were rooted on basal medium containing 1 mg/l NAA and were eventually transferred to soil. Chlorophyll variants were observed in about 6% of triticale cultures.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Kinetic studies on the diaqua form of cis-platin and various nucleobases

Kinetic studies on cis-[Pt(NH3)2(OH2)2]2+ and various nucleobases show that this ion reacts more quickly with guanosine than with adenosine, cytidine, and thymidine, and that a monophosphoric acid unit considerably enhances the rate of reaction of guanosine; the kinetic preference of 5'-GMP over 5'-AMP may point to a greater thermodynamic selectivity.     

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction

Background AimsViral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct.MethodsAssay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations.ResultsThe limit of detection was 10 copies/μL, whereas negative samples showed <1.3 copies/μL. Within and between assay imprecision coefficients of variation across the reportable range (10–10 000 copies/μL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R² >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations).ConclusionsDDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.